The effects of diphenylhydantoin on sodium transport in frog skin

Comp. gen. Pharmac., i97o , x, 365-37 t

365

THE EFFECTS OF D I P H E N Y L H Y D A N T O I N ON S O D I U M T R A N S P O R T IN FROG SKIN P A U L T. C A R R O L L AND J A M E S N. P R A T L E Y Department of Biological Sciences, San Jos6 State College, San Jost, California, U.S.A.

(Received 31 March, 197o) ABSTRACT I. The effect of dilute concentrations of diphenylhydantoin on the potential difference and short-circuit current of the isolated frog skin was studied. 2. The results indicated that the potential difference and short-circuit current were increased when the drug was put on the outside of the skin and they were decreased when the drug was put on the inside. 3. A qualitative comparison between the amount of S2Na pumped by unshorted control swatches exposed only to Ringer's solution on the outside corroborated the results obtained when diphenylhydantoin bathed the outside of the short-circuited skin. 4. The powerful duality of action exhibited by the drug was discussed in connexion with current models of frog skin ultrastructure.

DIPHENYLHYDANTOINsodium (dilantin) was introduced in 1938 as a drug for the treatment of epileptic seizures, including those of the grand mal and psychomotor types (Merritt and Putnam, 1938 ). Although it has had prominent use since then as an anticonvulsam drug, little is known of its cellular mode of action or its effects on ion movements across living membranes. However, one worker in I955 demonstrated that diphenylhydantoin induces an increased turnover of radio-sodium in the brain-cells of rats and that this turnover is not only accompanied by a decrease in intracellular sodium concentration but also a prevention of potassium loss to the bathing fluid (Woodbury, x955). The author interpreted these findings to indicate that the agent enhances the active extrusion of sodium by brain-cells and thereby lowers the intracellular brain sodium concentration. From these observations we postulated that diphenylhydantoin perhaps exerts its primary pharmacological action by affecting in a direct manner the general physiological mechanism of active sodium transport. To investigate this possibility the frog skin was

chosen as the experimental material because it has been characterized so well as a sodium pumping organ (see review by Ussing, i96o ). The fact that the tissue is electrically polarized, actively transports sodium, and contains a transport ATPase enzyme system which is found in other vertebrate tissue would support the validity of its use as a model system. Also, it is an ideal system for experimentation because of its ease of handling and ease of electrically monitoring the rates of sodium transport by the shortcircuit current technique (Ussing and Zerahn, I951). METHODS The skin of a frog (Rana p/p/ens) was excised from the abdomen and cut into symmetrical halves. One piece was used for measuring the electrical potential and the other for the shortcircuit current. Summer and early autumn frogs were used throughout these studies and were kept in a dark cold room for several days before sacririce. Each piece of skin was mounted between lucite chambers so that 2.84 sq. cm. of skin were exposed to aerated Ringer's solution comisting of io 5 mMNaC1, 5 mM KC1, and to mM NaHCO,. Calcium was not included in any of the solutiom because of its precipitating effect on the diphenyl-

366

CARROLL

AND

hydantoin test solution. The omission was justified on the basis of observations that frog skin is able to transport sodium normally for long periods when bathed in calcium-free Ringer's solution (Curran and Gill, x962). The electrical potential difference for both open-circuited and short-circuited swatches was measured by high-impedance electronic voltmeters and calomel electrodes which made contact with bathing solutions through agar bridges. The short-circuit current was continuously maintained across one swatch of skin by an external electromotive force through Ag-AgCI electrodes. Both swatches of skin were permitted to equilibrate Control

Test

Comp. gin. Pharmac.

PRATLEY

applied to the skins. In these sodium influx determinations, 0.33 gc. per ml. of tINa was added to test solutions, as well as control solutions, bathing the outsides of symmetrical open-circuited swatches from the same frog. Samples were removed from the inside bathing solutions t{ hours later. Triplicate counts were made of both control and test aliquots using a conventional G M end window tube. RESULTS

Fig. z is a typical r e p r e s e n t a t i o n o f the time-course o f the p o t e n t i a l difference a n d Control

Recovery

Tltt

Reco'~ery

t

30

A

i60

~30

120

o= r.,

20

,20 ~:

=" o

,o~

'10 .~

o 30 Short-circuit-~ control

60 Hlnutes Potential ~

90

difference

Fro. z.--Effect of diphenylhydantoin sodium on the potential difference and short-circuit current. During the control period the skin was bathed by Ringer's solution on both side*. During the test period the outside of the skin was bathed by a 8'0 × zo-' M diphenylhydantoin sodium Ringer's solution. During the recovery period the skin was bathed only by Ringer's solution. for 3° minutes before the introduction of the diphenylhydantoin test solution. Skin conductance was estimated from the short-circuit current and open-circuit potential difference (Rehm, x957). All bathing solutions utilized fell in the range o f p H 8.3+o.x and were at room temperature. The effects of diphenylhydantoin were noted for concentrations ranging from 2 . 4 x x o - * to 8. 4 × x o - ' M, concentrations which do not appreciably raise the oemolarity of Ringer's solution. Extensive statistical treatment of dosage effect was not undertaken became, as may be seen in the data summaries and as noted by other workers (see Pratley, 1965) , the electrical parameters of frog skins are highly variable in different animals. To corroborate by direct observations that the drug affects the sodium transport mechanism, experiments were also conducted in which " N a influx was observed when no external voltage was

Shoft-Orcuit ~ control

30 ~

~o Minutes 9o

,i'o

,~

Potential dtfferen
Fzo. 2.--Effect of diphenylhyclantoin sodium on the potential difference and short-circuit current. During the control period the skin was bathed by Ringer's solution on both sides. During the test period the inside of the skin was bathed by a 3.8 x zo-' M diphenylhydantoin sodium Ringer's solution alone.

short-circuit w h e n influenced b y a low c o n c e n t r a t i o n o f d i p h e n y l h y d a n t o i n on the e p i d e r m a l side o f the skin. T h e g r a p h shows t h a t a d d i t i o n o f d i p h e n y l h y d a n t o i n at the onset o f the test p e r i o d elicited a n i m m e d i a t e a n d significant elevation o f b o t h the p o t e n t i a l difference a n d short-circuit current. Both electrical p a r a m e t e r s r e a c h e d peaks w i t h i n 2 minutes o f the d r u g ' s i n t r o d u c t i o n a n d then d e c l i n e d at different rates until they reached plateaux. When diphenylhydantoin R i n g e r ' s solutions a r e r e m o v e d from the outside b a t h i n g c o m p a r t m e n t s a n d r e p l a c e d w i t h fresh R i n g e r ' s solution, a simultaneous decline occurs for b o t h the p o t e n t i a l difference a n d short-circuit current. Both electrical p a r a m e t e r s return, in the recovery p e r i o d , to values similar to those in the control period, i n d i c a t i n g t h a t the effects o f the d r u g ,

EFFECTS OF DILANTIN ON FROG SKIN

x97o , x

i n the c o n c e n t r a t i o n used in this experiment, were reversible. Table I is a s u m m a r y of the results of the I2 experiments i n w h i c h d i l a n t i n ' s effect o n the potential difference a n d short-circuit c u r r e n t was m e a s u r e d w h e n a p p l i e d to the

367

i n potential difference ( P < o . o o t ) , the increase i n short-circuit c u r r e n t (P< o.ooi), a n d the increase i n skin cond u c t a n c e ( P < o . o o l ) following d i p h e n y l h y d a n t o i n application are statistically significant.

Table / . - - E F F E C T OF DIPHENYLHYDANTOIN SODIUM ON THE OUTSIDE OF SKIN POTENTIAL

FROG No.

DIFFERENCE (mV.)

SHORT-CIRCUIT

CURRENT (gA.)

CONDUCTANCE

(mmhos per sq. cm.)

DILANTIN

C]ONCENTRATION (moles per litre)

x.R.L.* M.E.

t6

97

29

x98

2-I 2"3

2. R.L.

26

I15

t-6

M.E.

34

t65

1.7

3. R.L. M.E.

26 4°

4° 8o

o-54 0.70

8.0 x t o - '

4. R.L. M.E.

4°

16o

54

240

I' 5 I'6

4"8 X XO-~

5. R.L. M.E.

t6 19

42

58

0"97 I"4

4"8 x xo -4

6. R.L. M.E.

26 3a

x3o 195

1"76 2.x

4-8 x Io -4

7- R.L. M.E.

23 3°

65 lOG

l-o 1.2

4.6 x xo-6

8. R.L. M.E.

41 48

8o t5o

o'69 l.t

4.6 x I o - '

9. R.L. M.E.

32 38

I3o t8o

1"4 t.6

4"6 x l o - '

to. R.L. M.E.

33 4°

75 l Io

o.8o 0.97

4.6 x t o - '

t I.R.L.

20 23

I5O xSo

o'6

2.8

2.4 x to -s

t5

68 9°

1.6 1-8

8"4 x IO-'L

M.E. I2. R . L . M.E.

x8

B'O X IO -4

8"0 X I 0 -4

* R.L. refers to the resting level before dilantin application, and M.E. to the maximum effect or maximum stimulation. outside of the skin. T h e figures indicate that, for each of the 12 frogs, d i l a n t i n a p p l i c a t i o n resulted i n a n increase of b o t h the potential difference a n d short-circuit current. T h e use of a p a i r e d t-test showed t h a t the increase

Fig. 2 illustrates the typical effect oj d i p h e n y l h y d a n t o i n o n the potential difference a n d short-circuit c u r r e n t w h e n d i p h e n y l h y d a n t o i n was present o n the inside of frog skin. T h e g r a p h shows t h a t a d m i n i s t r a t i o n of

368

Comp. gen. Pharmac.

C A R R O L L AND P R A T L E Y

diphenylhydantoin at the beginning of the test period resulted in a rapid and sharp decline of both the potential difference and short-circuit current until they ultimately reached plateaux where they remain for the duration of the test period. Reversibility was not always observed in this series of experiments. Table H presents the summary of the results describing the effect of diphenylhydantoin on the potential difference and short-circuit current of frog skin when diphenylhydantoin was present on the inside. Potential difference results indicated that for each of the 7 frogs a decrease occurred upon addition of

Table II.--EFFECT OF POTENTIAL

FRoG No.

Dn~F~S~NCE (mY.)

skin is >o.oI).

statistically

significant

Fig. 3 illustrates a typical comparison between the effects of different concentration of diphenylhyclantoin when it is added to the outside of two symmetrical swatches of skin from the same animal. T h e higher concentration of diphenylhydantoin (4.67 x 10 -4 M ) gave rise to a greater increase in the short-circuit current generated by its swatch than the lower concentration of diphenylhydantoin (2.o x Io -~ M). T h e higher concentration of diphenylhydantoin typically elicited an immediate increase in the shortcircuit current followed by periodic decline

DIPHENYLHYDANTOIN SODIUM ON THE INSIDE OF SHORT-CIRCUIT CURRENT

(~tA.)

CONDUCTANCE

(mmhos per sq. cm.)

SKIN

DILANTIN CONCENTRATION

(moles per litre)

1. R.L.* M.E.

4°

82

0"73

33

96

1"0

2.

IO

35 25

1.2 i. 3

5"2 X IO -4

6o 52

0"44

5"2 X 10 -4

75 70

i. 4 3"3 4.2

5 .o X IO -4

R.L.

4"3 X IO - 4

M.E.

7

R.L. M.E.

48

R.L. M.E.

19

R.L. M.E.

14 8

133

R.L. M.E.

25

95 50

x. 3

4.6

II

7. R.L. M.E.

21

~6o

2'7

3"8 X 10 -4

8

5°

0'2

3. 4. 5. 6.

45 ,8

IOO

(o.o2>P

o.4 t 5"2 X 10 -4

I" 4

x

Io -4

x.6

* R.L. refers to the resting level prior to addition of dilantin and M.E. to the maximum effect or maximum inhibition. diphenylhydantoin. Results of the shortcircuit current measurements show that all but one of the skins manifested a decrease in short-circuit current resulting from diphenylhydantoin. T h e results of the paired t-test show that only the decrease in potential difference resulting from the presence of diphenylhydantoin on the inside of

until a plateau was reached. T h e lower concentration of diphenylhydantoin also resulted in an immediate increase in the short-circuit current, yet differed from the higher concentration in that it did not subsequently decline from its peak. This difference in behaviour was witnessed for most of the experiments which involved the

I97O ,

t

EFFECTS O F DILANTIN O N FROG SKIN

effect of diphenylhydantoin on the shortcircuit current. Table I I I presents the re.suits of the " N a experiments which were undertaken to corroborate the data presented in Table L Because the results in T a b l e 1 show that diphenylhydantoin does augment sodium transport across a short-circuited skin, it was thought that a determination of the dilantin effect on the transport of sodium across an unshorted skin through the use of ~SNa would offer a direct measure of the effect of dilantin on the sodium pump. According to the

369

the outside of skin, is a powerful stimulator of both the electrical potential and sodium transport. T h e fact that skin conductance is also elevated by diphenylhydantoin suggests that this drug m a y act by facilitating the passive entry of sodium into the skin. However, the facts that both the potential difference and short-circuit current are stimulated concomitantly and that the increase in skin conductance results from a proportional increase of the short-circuit current above its resting level greater than the increase of the potential difference above its resting level,

Table IlL--SoDIuM 22 INFLUX MZASURE~NTS

FRoo No.

4

BATHING

" N A INFLUX

SOLUTION*

(c.p.m.)

Dxt~tCnN (per cent change)

DILANTIN CONCENTRATION

(moles per litre)

Ringer's Dilantin

4o0 922

-J- i 3 o

2" 5

X IO -4

Ringer's Dilantin

265 331

+°5

2.6

x Io -~

Ringer's Dilantin

815 I743

+Ix3

'6

X IO -4

Ringer's Dilantin

IoSI

Ringer's Dilantin

813

Ringer's

7

EFFECT OF

755

I

+43

2.6 × Io-'

+44

2"6 × xo-'

+46

2"0 X 10 -4

+35

2"0 × I 0 - *

564 587

Dilantin

856

Ringer's Dilantin

884

654

* Swatches were either bathed only with Ringer's solution during the course of each experiment or bathed by dilantin Ringer's solution on the outside during the test period. results in Table I I I all 7 skins tested showed a greater amount of 22Na transport across swatches bathed on the outside by Ringer's solution a n d this difference was statistically significant (o.o2>P> 0"OI).

DISCUSSION T h e results of the present study indicate that diphenylhydantoin, when applied to

indicate that skin conductance m a y not be the explanation for the observed effects. T h e effects of diphenylhydantoin on the inside are also pronounced but, surprisingly, the drug acts as a powerful inhibitor of both the electrical potential and sodium transport. In this case the drug has no consistent effect upon skin conductance. Perhaps the most reasonable assumption concerning the duality of action is that diphenylhydantoin, whether

CARROLL

37 °

AND

present on the inside or outside of the skin, acts on the same factor associated with the active transport mechanism. I f the frog skin ultrastructure ( F a r q u h a r a n d Palade, t964) a n d the transport A T P a s e gradients in the epithelial layers o f the frog skin ( F a r q u h a r and Palade, ]966; Dahl a n d Pratley, i967) are considered, the following speculation m a y be offered to explain h o w an inhibitory effect of the d r u g might arise. W h e n a d d e d to the inside bathing solution, an inhibition of active sodium transport w o u l d result if dilantin stimulated the transport across the level of epithelial cells closest to the inside bathing solution. Such a stimulation would enable more sodium to enter the intercellular Control

Test

PRATLEY

Comp. gen. Pharmac.

in other types of effects observed on frog skin. For example, low concentrations of magnesi u m ions, sufficient to inhibit histochemicaUy the transport A T P a s e enzyme, when applied to the outside of the skirt inhibit sodium influx; yet when the divalent ion is applied to the inside of the skin, stimulation sometimes occurs (Curran a n d Gill, I962; Dahl a n d Pratley, z967). While these data strongly suggest that dilantin has a marked effect u p o n the sodium transport mechanism, no specific site of action is revealed. W h e t h e r it affects the transport enzyme directly, its substrate, or some cofactor cannot be stated at this time.

Recovery

ACKNOWLEDGEMENTS I

This work was supported partly by funds from The National Institute of Health (GM-o7872) and the San Jos6 State College Foundation.

~0 ~

I00.

o

3o

~o

9o

,'~o

Minutes Hi|h Low concentr~lon ; = ~ ¢oae~mteaUon ± ~ -"

Fio. 3.--Effect of diphenylhydantoin sodium concentration on the short-circult current of frog skin. During the control period both swatches from the same animal were bathed by Ringer's solution. During the test period one swatch was bathed by 2-2 x i o - ' M diphenylhydantoin sodium Ringer's solution on the outside while the other was bathed by 4-67 x to -4 M diphenylhydantoin sodium Ringer's solution on the outside. During the recovery period both swatches are again bathed only by Ringer's solution. channels closest to the inner b a t h i n g compartment, thus opposing the normal gradient o f passive sodium diffusion from the outer intercellular channels towards the inner ones. This ' b a c k diffusion' w o u l d decrease the potential difference a n d sodium influx. A l t h o u g h this explanation is conjectural, this duality of action m a y have a counterpart

REFERENCES CURRAN, P. F., and GILL, J. (1962), ' The effect of calcium on sodium transport by frog skin ', 07. gen. Physiol., ~ 625--64i. DAm., R. H., and P a x ~ Y , J. N. (x967) , ' The effects of magnesium on nucleoside phosphatase activity in frog skin ', 07. Cell Biol., 33, 41 t-4x8. FARQ.UHAR, M. G., and P A L A D E , G . E . (x964) , ' Functional organization of amphibian skin ', Proc. hath. Acad. Sci. U.S.A., 5x, 569-577 . (1966), ' Adenosine triphosphatase localization in amphibian epidermis ', 07. Cell Biol., $o, 359-379. MEm~rr, H. H., and P t r r s ~ , T. J. (t938), 'Sodium diphenylhydantoinate in the treatment of convulsive disorders ', 07. Am. meat. Ass., sxs, so68-qo72. P~TX~Y, J. N. (t965), ' Comparative studies on the electrical properties and enzyme activities in skim of Rana ¢atesbeiana ', Comp. Biochem. Physiol., i t , 56x-57L REmd, W. S. (1957), ' Electrical resistance of the stomach ', in Metabolic Aspects of Transport across Cell Membranes (ed. M m ~ n ~ , Q. R.), pp. 322-329 . Madison, Wis.: University of Wisconsin Press. U~SINO, H. H. (t96o), ' The alkali metal ions in isolated systems and tissues ', in The Alkali Metal Ions in Biology, pp. x-i 28. Berlin: SpringerVerlag.

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EFFECTS OF DILANTIN ON FROG SKIN

USSING,H. H., and ZERAHN,K. ([95]), ' Active transport of sodium as the source of electric current in the short-circuited isolated frog skin ', Acta physiol, stand., 23, t xoI27. WOODBURY, D. M. (t955), 'Effect of diphenylhydantoin on electrolytes and radio-sodium turnover in brain and other tissues of normal

37 I

hypnoatremic and po6tictal rats ', 07. P/tarmac. exp. Ther., ix5, 74-95-

Key Word Index: Frog skin, dilantin, active ion transport, epithelial membrane ion transport, bioelectric potentials, sodium pump.